An
array
of
gel-coated
ECG
electrodes
(Red
Dot;
3
M,
St.
Paul,
MN)
are
embedded
in
the
platform
floor
and
spaced
approximately
3
cm
in
between
each
other
such
that
forelimb
and
hindlimb
paws
can
simultaneously
come
in
contact
with
the
electrodes.
Filter
papers,
with
openings
for
the
electrodes,
are
used
to
prevent
mouse
urine
from
short-circuiting
the
signals.
The
electrodes
are
connected
to
an
amplifier
(HP78901A,
Hewlett-Packard,
Andover,
MA)
by
a
shielded
3-electrode
lead
set
(M1605A
Snap,
Hewlett-Packard,
Andover,
MA).
The
signals
are
digitized
with
16-bit
precision
(DI-220,
DATAQ
Instruments,
Inc.,
Akron,
OH)
at
a
sampling
rate
of
2500
samples/s.

Data
are
transmitted
to
the
mousespecifics.com
web
site
(Mouse
Specifics,
Inc.,
Boston,
MA)
using
standard
file-transfer
protocols
for
ECG
signal
analyses
by
e-MOUSE®.
e-MOUSE®,
an
internet-based
physiologic
waveforms
analysis
portal,
incorporates
Fourier
analyses
and
linear
time-invariant
digital
filtering
of
frequencies
below
2Hz
and
above
100Hz
to
minimize
environmental
signal
interferences
during
ECG
waveform
processing.
The
software
plots
its
interpretation
of
P,Q,R,S,
and
T
for
each
heart
beat
(see
below);
so
that
spurious
data
resulting
from
unfiltered
noise
or
motion
artifacts
may
be
rejected.

A
10
min
period
of
acclimatization
is
implemented
when
using
the
non-invasive
ECG
screening
system
because
the
novelty
of
the
environment
may
induce
a
state
of
anxiety
in
conscious
mice
(manifested
by
body
tremors,
shivering,
and
general
increase
in
activity,
all
of
which
may
introduce
electrical
signals
that
confound
acquisition
of
ECG).
Two
mice,
one
placed
on
each
side
of
the
tower
platform,
are
acclimated
at
the
same
time.
As
the
mice
become
acclimated
to
the
test
conditions,
they
tend
to
become
more
relaxed,
and
more
likely
to
contact
the
electrode
pads,
which
promote
recordable
ECG
signal.

Procedure:
Electrocardiograms
(ECG)
in
conscious
mice
using
the
AnonyMouse
ECG
screening
system

To
measure
electrocardiographic
(ECG)
parameters
in
conscious,
unanesthetized,
and
unrestrained
11wk-old
mice,
the
AnonyMouse
ECG
screening
system
is
used.
The
success
of
obtaining
recordable
data
depends
upon
properly
acclimatizing
the
mice
to
the
testing
conditions,
and
properly
positioning
the
paws
to
be
in
full
contact
with
the
electrodes.

Staging
mice
for
ECG
recording
a.
Mice
are
gently
removed
from
their
cages,
picked
up
by
their
tails,
and
propped
on
the
ECG
platform
(see
Figure
3
above)
with
the
least
amount
of
provoked
anxiety
and
stress
and
then
allowed
to
acclimate
for
10
min.b.
An
acclimated
mouse
is
then
gently
prodded
and
positioned
at
the
center
of
the
platform
such
that
the
paws
come
in
contact
with
the
three
electrodes
using
a
small
wooden
stick
with
a
cotton
swab
on
one
end.
The
two
front
paws
and
left
hind
paw
in
contact
with
the
lead
plates
with
the
mouse
standing,
not
sitting,
are
optimized
testing
conditions.
c.
The
ECG
system
is
then
turned
ON,
started
and
operated
according
to
manufacturer's
protocol.
While
waiting
for
signals
to
appear,
another
mouse
is
gently
placed
on
the
vacated
side
of
the
platform
to
acclimate.
d.
The
output
from
the
amplifier
is
scrutinized
carefully
such
that
only
data
from
a
continuous
display
of
20-30
ECG
signals
or
waveforms
(in
2-3s)
are
recorded
and
used
in
the
analyses
(see
Figure
4
below).

Figure
4.
Examples
of
recorded
mouse
ECG
signals.
Fig.
4A.
ECG
signals
from
a
conscious
or
unanesthetized
mouse.
Note
the
difficulty
in
determining
the
exact
beginning
and
ending
of
the
T
wave.
Fig.
4B.
ECG
signals
from
an
anesthetized
mouse.

e.
At
the
end
of
each
session,
the
tower
and
platform
is
disassembled
by
its
various
parts
(shields,
platform,
lead
plate)
for
cleaning
using
mild
detergent
and
paper
towels,
and
then
disinfected
and
allowed
to
dry
with
alcohol.
The
lead
plate
wherein
the
electrodes
are
attached
are
kept
and
not
thrown
away,
while
the
gel-coated
ECG
electrodes
are
periodically
changed
and
replaced.f.
The
cleaned
platform
is
assembled
for
the
next
session.
A
piece
of
paper
towel
is
placed
beneath
the
back
shield
and
on
each
sidewall
of
the
platform
to
protect
it
from
soiling.
The
distance
of
the
back
shield
to
the
platform
is
adjusted
according
to
the
size,
such
that
the
closest
channel
to
the
platform
is
used
for
small
to
medium
sized
mice,
and
one
channel
farthest
back
is
used
for
larger
mice.

ECG
data
analyses
and
definitions
using
e-MOUSE®

Figure
5.
ECG
waveforms.

a.
Heart
rate
is
calculated
using
the
number
of
R-wave
peaks.b.
Heart
rate
variability
is
calculated
as
the
average
of
the
differences
between
a
given
series
of
heart
rates
from
a
set
of
ECG
signals
or
waveforms.c.
QRS
complex
is
automatically
determined
from
the
beginning
of
a
Q-wave
peak
to
the
end
of
the
S-wave
peak.d.
QT
interval
is
defined
as
the
segment
in
ECG
waveform
beginning
at
the
peak
of
the
Q-wave,
including
the
inverted
and/or
biphasic
portions
of
the
T-wave,
and
ending
at
the
peak
of
the
T-wave.e.
The
end
of
the
T-wave
of
each
ECG
waveform
is
defined
as
the
point
where
the
T-wave
returns
to
baseline
or
isoelectric
line.f.
Isoelectric
line
or
baseline
is
defined
as
the
average
voltage
between
the
preceding
P-wave
and
the
QRS
interval.g.
The
QT
intervals
are
heart
rate
(RR)
corrected
(QTc)
by
application
of
the
equation
recommended
by
Mitchell
et
al.
for
use
in
conscious
mice:
QTc
=
QT/SQRT(RR/100)h.
For
additional
information: